The mechanisms by which human breast carcinomas acquire resistance to antiestrogen therapy remain incompletely understood. In an unbiased search for genes which regulate this process, Dorssers et al determined that upregulation of either a novel protein, BCAR3, or the focal adhesion adapter protein p130Cas are reproducible genetic events that induce such antiestrogen resistance. In concurrent and independent work, we cloned murine AND-34/BCAR3 and demonstrated that it associates with p130Cas through a Cdc25 GEF-like domain. Using Western analysis, we find that AND-34 is expressed at high levels in a subset of human breast carcinomas but not normal breast tissue. We subsequently determined that overexpression of AND-34 induces PI3K, Akt and Rac activation in breast cancer cell lines. Specific Aims: 1) To determine the mechanism by which overexpression of AND-34 in human breast cancer cells induces PI3K activation, we will: A) Examine whether AND-34 associates with and positively regulates PI3K activation by transmembrane tyrosine kinase receptors in breast cancer cell lines. This work will include an analysis of signaling in AND-34 -/- tissues; B) Examine the role of p130Cas in AND-34-mediated PI3K activation; C) Examine the role of Src kinases in such PI3K activation. 2) To determine how AND-34 and related gene family members affect PI3K signaling and clinical outcome in primary human breast cancers and to examine the effect of AND-34 on ER signaling, we will: A) Contrast the ability of human NSP1, AND-34 and NSP-3 to induce Akt and Rac activation and antiestrogen resistance in human breast cancer cell lines. B) Determine by Western analysis whether NSP1, AND-34 or NSP-3 expression in primary human breast cancer specimens correlates with PI3K activation or ERa expression; C) Determine by immunohistochemistry whether NSP1, AND-34 or NSP-3 expression in primary human breast cancer specimens correlates with ERa expression, disease-free or overall survival. D) Determine if AND-34 overexpression induces ligand-independent ERa-mediated ERE transactivation.